The present invention relates to a modified chitin binding domain and methods for making the same where the modification alters the properties of the chitin binding domain so that it becomes capable, under select conditions, of elution from a substrate for which it has specific affinity.
Although a number of different approaches to protein purification exist, the application of recombinant techniques to generating fusion proteins from target proteins and substrate binding proteins (affinity tags) has provided efficient methods of separating target proteins from complex mixtures and/or large volumes. (LaVallie and McCoy, Curr. Opin. Biotechnol., 6:501–506 (1995), U.S. Pat. Nos. 5,834,247 and 5,643,758). Examples of substrate binding proteins include the chitin binding domain (CBD or ChBD) of chitinase which binds chitin substrate (U.S. Pat. No. 5,837,247, Xu et al., Methods Enzymol. 326:376–418 (2000)), maltose binding protein which binds an amylose substrate (U.S. Pat. No. 5,643,758), cellulose binding domain from cellulase which binds cellulose (U.S. Pat. Nos. 5,962,289; 5,928,917; and 6,124,177) and His-Tag (an oligopeptide) which binds a Nickel charged column. (Van Dyke, et al. Gene 111:99–104 (1992)). In addition to the above, Glutathione S-transferase (GST) bind sepharose TM4B resin (Smith, D. B., and Johnson, K. S. Gene 67:31–40 (1998)).
Each of the above affinity tags has certain limitations. For example, CBD irreversibly binds to chitin substrate and cannot be eluted under non-denaturing conditions. However, CBD represents a potentially useful affinity tag with widespread application since it is readily obtained from any of a family of enzymes identified as chitinases that are capable of hydrolyzing chitin. As might be expected, chitinases are produced by a diverse range of organisms that either contain chitin or rely on chitin as a food source. These organisms include bacteria, fungi, plants and vertebrates. (Watanabe et al. J. Bacteriol., 176:4465–4472 (1994), Jolles et al., Chitin and Chitinases, Birkhäuser Verlag, Basel (1999); Hashimoto et al., J. Bacteriol. 182:3045–3054 (2000)). CBD binds to chitin, a polysaccharide abundantly represented in nature. It is found in many fungal cell walls, nematode and insect exoskeletons, and crustacean shells.
Chitinases are characterized by a chitin binding domain (CBD) and a catalytic domain. For example, Chitinase A1 which is produced by Bacillus circulans WL-12 contains three discrete functional domains: an N-terminal family 18 catalytic domain, a tandem repeat of fibronectin type III-like domains and a C-terminal chitin-binding domain (FIG. 1) (Watanabe et al., supra (1994)). Moreover, since CBD is located within the chitinase at a site that is distinct from the catalytic domain, it naturally lacks hydrolytic activity when isolated from the enzyme for use as an affinity tag.
While CBD has a number of useful properties it lacks the property of reversible binding to chitin under non-denaturing conditions which limits its general utility.